利用绿色荧光蛋白优化辣椒遗传转化体系

【目的】农杆菌介导的辣椒遗传转化较为困难，至今仍未建立高效转化体系。绿色荧光蛋白(GFP)基因是植物遗传转化中常用的报告基因，以GFP为报告基因，优化农杆菌介导的辣椒遗传转化体系。【方法】利用GFP表达系统，统计3个辣椒品种(‘HP’‘8214’和‘L55’)中3种不同外植体(子叶、下胚轴和Flamingo-bill外植体)的不定芽分化率、不定根分化率与荧光阳性率，探究农杆菌侵染浓度、侵染时间、预培养时间和共培养时间等因素对不定芽、不定根分化率及荧光阳性率的影响。【结果】3个辣椒品种的Flamingo-bill外植体不定芽分化率均显著高于下胚轴与子叶外植体，其中‘L55’ Flamingo-bill外植体的不定芽分化率最高、达77.59%，故选用‘L55’ Flamingo-bill外植体进行后续研究。4种农杆菌不同侵染浓度和时间组合下，‘L55’Flamingo-bill外植体均可产生不定芽、不定根及表达GFP的愈伤组织，当农杆菌侵染浓度为OD₆₀₀=0.05，侵染时间为30 min时，不定芽分化率和荧光阳性率最高，分别达48.39%、4.84%。在6种不同预培...

Optimization of Genetic Transformation System of Peppers by Green Fluorescent Protein

【Objective】The genetic transformation of pepper mediated by Agrobacterium was a very difficult process, and no efficient transformation system had been established so far. Green fluorescent protein (GFP) gene is a common reporter gene in plant genetic transformation. In the study, GFP was used as the reporter gene to optimize the Agrobacterium-mediated genetic transformation system of pepper. 【Method】By using GFP expression system, three types of explants (cotyledons, hypocotyls and Flamingo-bill explants) from 3 pepper varieties (‘HP’ ‘8214’ and ‘L55’) were counted according to the adventitious bud differentiation rate, adventitious root differentiation rate and fluorescence positive rate. The effects of factors including infection concentration and infection time, pre-culture time and co-culture time on adventitious bud differentiation rate, adventitious root differentiation rate and fluorescence positive rate were explored.【Result】The adventitious bud differentiation rate of Flamingo-bill explants was significantly higher than that of the hypocotyl and cotyledon explants, among which the ‘L55’ Flamingo-bill explant had the highest adventitious bud differentiation rate of 77.59%. Therefore, the ‘L55’Flamingo-bill explant was selected for subsequent research. Under 4 different combinations of Agrobacterium infection concentration and infection time, all of them could produce adventitious buds, adventitious roots and GFP-expressing calluses. When the infection concentration of Agrobacterium was OD600=0.05 and the infection time was 30 min, the adventitious bud differentiation rate and fluorescence positive rate of Flamingo-bill explants were the highest, reaching 48.39% and 4.84%, respectively. Under 6 different pre-culture and co-culture time combinations, the Flamingo-bill explants also produced adventitious buds and adventitious roots, and the highest adventitious bud differentiation rate and fluorescence positive rate were 48.44% and 12.50%, respectively, under the treatment of pre-culture for 1 d and co-culture for 1-2 d. Finally, PCR detection of adventitious roots and calluses with GFP fluorescence expression showed that the 3 target genes GFP, Kan and Cas9 all presented signal in the fluorescence positive tissues, indicating that the T-DNA insertion mediated by Agrobacterium was successful and the transformation was stable.【Conclusion】The explant type of pepper, Agrobacterium infection concentration, infection time, pre-culture time and co-culture time all have effects on the genetic transformation efficiency of pepper. Screening appropriate transformation conditions with GFP as a reporter gene could improve the efficiency of Agrobacterium-mediated genetic transformation of pepper.